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Determination of 2-acetylaminofluorene and its metabolites in urine by high pressure liquid chromatography
BIOCHEMICAL
MEDICINE
16, 95-103 (1976)
Determination of 2-Acetylaminofluorene and Its Metabolites Urine by High Pressure Liquid Chromatography’ FLOYD Department of Health, The National Center
R. FULLERTON Education and for Toxicological
in
AND C. D. JACKSON Welfare, Food and Drug Administration, Research. Jefferson, Arkansas 72079
Received April 20, 1976
2-Acetylaminofluorene (2-AAF)2 has been used as a model chemical carcinogen since its tumorigenic activity was discovered by Wilson et al. (1) in 1941.2-AAF has been shown to produce tumors in numerous tissues of the rat including liver, Zymbal’s gland, and mammary tissue (l-3) and is known to be a potent bladder carcinogen in the mouse (4). 2-AAF is inactive as a carcinogen per se and requires metabolic activation to the N-hydroxy metabolite (5). The metabolism and reactivity of N-OH-AAF was recently reviewed by Weisburger and Weisburger (6). The other major hydroxy metabolites found in the urine following administration of 2-AAF are I-OH-AAF, 3-OH-AAF, SOH-AAF, and 7-OH-AAF (7,8). Earlier studies on the urinary excretion of 2-AAF and its metabolites utilized paper, silicic acid column, or thin-layer chromatography (7,s). These methods of fractionating and quantitating the various metabolites are time consuming and lack the desired sensitivity for detailed metabolism studies. Recently, Irving (9) reported that N-hydroxy-N-arylacetamides could be analyzed by gas-liquid chromatography as their trimethylsilyl derivatives. Subsequently, Gutmann (10) reported a method utilizing high pressure liquid chromatography for fractionating 2-AAF and some of its hydroxylated metabolites formed by an in vitro microsomal system. We have investigated both gas chromatography and high pressure liquid chromatography as possible methods for analyzing urine samples for 2-AAF and its hydroxy metabolites. It was found that ether extracts of urine contained interfacing materials that precluded the application of the ’ This paper was presented, in part, at the Thirteenth Annual Meeting of the Society of Toxicology, Washington, D.C., March 12, 1974. ’ Abbreviations used: 2-AAF, 2-acetylaminofluorene (Chem. Abstr. nomenclature, N-2fluorenylacetamide); 2-AF, 2-aminofluorene: N-OH-AAF, N-hydroxy-2-acetylaminofluorene; I-OH-AAF, 3-OH-AAF, S-OH-AAF, and 7-OH-AAF, the respective ring-hydroxy derivatives of 2-AAF. 95 Copyright All rights
@ 1976 by Academic Press. Inc. of reproduction in any form resewed.
96
FULLERTON
AND
JACKSON
published gas or liquid chromatographic procedures to the analvsis of urine samples. There was, therefore, a need to modify the existing methods or to develop a new one to permit the analysis of urine samples. This report presents the results of a study in which a high pressure liquid chromatographic method was developed to identify and quantitate 2-AAF and its major metabolites in urine samples. MATERIALS
AND METHODS
Standards of l-OH-AAF, 3-OH-AAF, S-OH-AAF, 7-OHAAF, N-OH-AAF, and 2-AAF were obtained from Dr. Charles Irving, Veterans Administration Hospital, Memphis, Tennessee. Identity and purity of each standard was confirmed by uv spectroscopy (11,12) and thin-layer and paper chromatography (8). The diethyl ether, acetonitrile and heptane were Nanograde, all-glass-distilled solvents from Mallinckrodt Chemical Works, St. Louis, Missouri. The ether and heptane were stored over barium oxide prior to use. Isolation of urinary metabolites. Urinary metabolites were isolated as described by Weisburger et al. (8). Urine samples were mixed with l/10 volume of 1 M sodium acetate, pH 6, and extracted with ether to remove 2-AAF and unconjugated metabolites. The aqueous layer was incubated with fi-glucuronidase (400 units/ml) for 2 hr at 37°C to hydrolyze glucuronide conjugates. Metabolites freed by the P-glucuronidase were extracted with ether and combined with the previous extract. For the purpose of this report, the sulfate conjugates remaining in the aqueous layer were discarded. The ether was evaporated with nitrogen, and the residue was redissolved in the mobile phase described for liquid chromatography. Liquid chromatography. A Waters 2021401 chromatograph equipped with two Model 6000 pumps and a Model 660 solvent programmer was used. The detector utilized a uv photometer operated at 280 nm. Two high pressure liquid chromatography systems were used. The first was essentially the same as described by Gutmann (10). Samples dissolved in 10% heptane in diethyl ether were injected into a column (4 ft x 2-mm i.d.) packed with Carbowax 400 on Porasil C (Waters Associates, Milford, Massachusetts). Fractions were then eluted with 10% heptane in diethyl ether at a rate of l-2 ml/min. The second system consisted of a column (2 ft x 2-mm i.d.) packed with C,,/Corasil (Waters Associates) with a mobile phase of acetonitrile:water using a step-flow gradient. For the first 4 min, a flow of 0.3 mI/min of 12% acetonitrile in water was maintained. After 4 min, a second pump was engaged which added 0.3 ml/min of 60% acetonitrile in water to the initial flow. Chemicals.
CHROMATOGRAPHY
OF Z-AAF METABOLITES
97
RESULTS Liquid Chromatography
Fractionation of 2-AAF and its hydroxy metabolites on a 4-ft Carbowax 400 column is shown in Fig. 1. Each metabolite was identified by comparison of the retention time with that of a corresponding standard. Our results confirmed Gutmann’s report (10) in that N-OH-AAF, SOH-AAF, and 7-OH-AAF were each separated on the Carbowax 400 column. However, we found I-OH-AAF and 3-OH-AAF to have approximately the same retention time as 2-AAF on this column. Although separation of SOH-AAF and 7-OH-AAF was incomplete on the 4-ft column, they could be separated by using a longer column. A more efficient separation of 2-AAF and the ring-hydroxy metabolites by liquid chromatography is shown in Fig. 2. 2-AAF, I-OH-AAF, 3-OH-AAF, S-OH-AAF, and 7-OH-AAF were each well resolved by reverse phase liquid chromatography on a 2-ft column of C&orasil, using acetonitrile:water as the mobile phase. N-OH-AAF, however, was not eluted from this column. The linearity of response (peak height x attenuation) of the chromato-
t
0
:
:
4
:
o
li
TIME
:
’
li (MU)
:
:
16
:
I
20
FIG. 1. Elution profile of 2-AAF and hydroxy metabolites separated by liquid chromatography on a 4-ft x 2-mm i.d. column packed with Carbowax 400. The mobile phase was 10% heptane in diethyl ether with a flow rate of 1.3 mUmin.
98
FULLERTON
AND JACKSON
; Y P--f-i-+ 0
4
! I
: : II 16 TIME (MN)
:
: 20
: 44
FIG. 2. Elution profile of 2-AAF and hydroxy metabolites separated by liquid chromatography using a 2-ft by 2-mm i.d. stainless-steel column packed with C,&orasil. The mobile phase was acetonitrile:water using a step-flow gradient. For the first 4 min, a flow of 0.3 ml/min of 12:88 acetonitrile:water was maintained. At 4 min, a second pump was engaged which added 0.3 mUmin of 60140 acetonitrile:water to the initial flow.
graphic system to the various metabolites is shown in Fig. 3. The response to N-OH-AAF on the Carbowax 400 column was linear from approximately 100 ng to 4.0 pg, while the response to 2-AAF on the C,,/Corasil column was linear from 12 ng to 2 pg (Fig. 3a). Figure 3b shows that the response to each of the ring-hydroxylated metabolites was linear from approximately 10 ng to 2 pg. 5-OH-AAF produced the same response as ‘I-OH-AAF while the response to l-OH-AAF and 3-OH-AAF were identical. Analysis of Urine Samples
Samples of mouse urine containing standards were extracted as described above and the extracts were fractionated by liquid chromatography. N-OH-AAF was eluted from the Carbowax 400 column in fractions relatively free of interfering urinary components and could be quantitated directly from the Carbowax 400 column. There were, however, interfering urinary components when ether extracts of urine were run on C,,/Corasil column. Figure 4a shows the elution profile of an ether extract of control urine chromatographed on the C,,/Corasil column. Numerous components were present which would interfere with iden-
CHROMATOGRAPHY
OF 2-AAF METABOLITES
99
FIG. 3. Linearity of response obtained by liquid chromatography expressed as peak height (absorbance at 280 nm) vs sample size (nanograms). (a) N-OH-AAF determined on a Carbowax 400 column; 2-AAF determined on C,,/Corasil column. (b) Ring-hydroxy derivatives determined on a C,$Corasil column.
tification and quantitation of 2-AAF and the ring-hydroxy metabolites. Subsequently, it was found that the urinary components had markedly different retention times on Carbowax 400 and C,,/Corasil columns. An ether extract of control urine was fractionated on a Carbowax 400 column and fractions corresponding to retention times of 2-AAF and the ringhydroxylated metabolites (8-18 min, Fig. 1) were pooled, concentrated, and rechromatographed on the C,,/Corasil column. The results, shown in Fig. 4b, indicated that the urinary components normally eluted from the C&orasil column were largely removed by a prior fractionation on a
100
FULLERTON
AND JACKSON
FIG. 4. Liquid chromatography of mouse urine components on a C,,/Corasil column. (a) Ether extract of control mouse urine chromatographed on a C,,/Corasil column with no previous cleanup. (b) Same extract was run on a Carbowax 400 column and fractions corresponding to 8 to 18 min (Fig. 2) were concentrated and run on the C&orasil column.
Carbowax 400 column. A combination of the two columns thus provided a method for resolving and quantitating each of the compounds. In a routine analysis, an ether extract of urine was chromatographed on the Carbowax 400 column, and the N-OH-AAF was determined directly from the elution profile. Fractions containing 2-AAF and the ring-hydroxy metabolites were collected, concentrated, and chromatographed on the C&orasil column. Each metabolite was then determined from the elution profile. An analysis of an actual urine sample is shown in Fig. 5. Female BALB/c mice were maintained on a diet containing 0.05% AAF for 2 weeks, and urine was collected overnight in a metabolism cage. Urinary metabolites were isolated as described above. The ether extract was concentrated and fractionated on the Carbowax 400 column (Fig. Sa). Fractions eluting between 13 and 26 min were concentrated and chromatographed on the C,,/Corasil column (Fig. 5b). The quantity of each metabolite was determined by comparing its peak height with that of a standard. The recovery of each metabolite from urine was determined. A mixture containing 200-400 ng of each metabolite was added per milliliter of control urine and processed as described in Materials and Methods. The results are presented in Table 1. The recovery of N-OH-AAF was determined from the Carbowax 400 column while that of 2-AAF and the ring-hydroxy metabolites were determined from the C,,/Corasil column.
CHROMATOGRAPHY
OF 2-AAF METABOLITES
101
FIG. 5. Liquid chromatography profiles of ether extracts of urine from female BALBlc mice maintained on a diet containing 0.05% 2-AAF for 2 weeks. (a) Profile obtained on Carbowax 400 column. (b) Profile of fractions 13 to 26 min from Carbowax 400 column concentrated and run on C,,/Corasil column. Conditions of chromatography described in Materials and Methods.
Recovery of N-OH-AAF proved to be low; approximately 20% was consistently lost during the extraction and/or chromatography procedures. DISCUSSION 2-AAF has been used not only as a model chemical carcinogen but is presently used as a positive control in numerous laboratories in the bioassay of potential carcinogens. Therefore, there is a need for a rapid, sensitive analytical method for determining the amounts of 2-AAF and its major metabolites in urine. Our investigations indicated that the gas chromatographic method described by Irving (9) provided an excellent method for analyzing mixtures of 2-AAF and its hydroxy metabolites when isolated from a relatively “clean” system, but was not applicable to urine samples because of interfering urinary components. Gutmann (10) reported that 2-AAF, N-OH-AAF, S-OH-AAF, and 7-OH-AAF were resolved by liquid chromatography on Carbowax 400 columns, but that l-OH-AAF and 3-OH-AAF had the same retention time as the parent compound 2-AAF. Our results (Fig. 1) confirm his report. However, when the method was applied to ether extracts of urine, numerous urinary components were present which interfered with the determination of several 2-AAF metabolites. Further studies indicated,
102
FULLERTON
AND JACKSON
TABLE RECOVERIES
OF
2-AAF
WHEN
AND
ANAI.YZED
HYDROXY
I METABOLITES
BY LIQUID
Metabolite
FROM
Moust-
URINE
CHROMATOGRAPH\
Percentage recovered”
2-AAF l-OH-AAF 3-OH-AAF .5-OH-AAF 7-OH-AAF N-OH-AAF
101 2 90 k 100 k 112 2 114 k 79 +
2.50 7.3 8.5 6.3 9.3 3.7
Metabolites were added to control urine at concentrations of 200-600 rig/ml. Recoveries of 2-AAF and ring-hydroxy metabolites were determined from a C,,/Corasil column, while recovery of N-OH-AAF was determined from a Carbowax 400 column. * r SD, N = 4.
however, that the interfering urinary components &d different retention times on Carbowax 400 and C,,/Corasil solumns. A combination of the two columns was then found to provide an excellent method for resolving and quantitating 2-AAF, N-OH-AAF, l-OH-AAF, 3-OH-AAF, S-OHAAF, and 7-OH-AAF in the presence of urinary components. The response of the system to each of the metabolites was linear over a range of 10 ng to 2 lug with the exception of N-OH-AAF .which deviated from linearity below 100 ng (Fig. 3). Recoveries of each metabolite from urine were good except for N-OH-AAF. Approximately 20% of this metabolite was consistently lost during the analytical process. This procedure provides a rapid and sensitive analytical method for determining the amounts of 2-AAF and its major metabolites in urine of experimental animals. SUMMARY
2-Acetylaminofluorene, a well-documented carcinogen, is used extensively as a positive control in the bioassay of numerous suspect carcinogens. Consequently, there is a need for a rapid, sensitive analytical method for determining 2-acetylaminofluorene and its metabolites in urine. Such a method would be useful in monitoring workers for potential exposure as well as for determining the applicability of the test species by metabolism studies. A high pressure liquid chromatography method for the identification and quantitation of 2-acetylaminofluorene and its major metabolites in urine has been developed. The primary obstacle in developing the method was the presence of large amounts of endogenous urinary components in ether extracts of urine having chromatographic properties similar to
CHROMATOGRAPHY
OF 2-AAF METABOLITES
103
2-acetylaminofluorene and its metabolites. This problem was obviated by the use of a combination of Carbowax 400 and C,,/Corasil columns. N-Hydroxy-2-acetylaminofluorene was determined directly from the Carbowax 400 column. Fractions containing 2-acetylaminofluorene and its l-, 3-, 5, and 7-hydroxy metabohtes were then separated from interfering materials and quantitated by fractionation on a C,,/Corasil column. The response of the system was linear down to 100 ng of N-hydroxy-2acetylaminofluorene and to 10 ng of 2-acetylaminofluorene and the other metabolites. REFERENCES 1. Wilson, R. H., De Eds, F., and Cox, A. J., Cancer Res. 1, 595 (1941). 2. Irving, C. C., Janss, D. H., and Russell, L. T., Cancer Res. 31, 387 (1971). 3. Gutmann, H. R., Malejka-Giganti, D., Barry, E. J., and Rydell, R. E., Cancer Res. 32, 1554 (1972). 4. Wood, M., Eur. J. Cancer $41 (1969). 5. Miller, J. A., and Miller, E. C., Progr. Exp. Tumor Res. 11, 273 (1969). 6. Weisburger, J. H., and Weisburger. E. K., Pharmacol. Rev. 25, 1 (1973). 7. Weisburger, J. H., Weisburger, E. K.. Morris, H. P., and Sober, H. A., J. Nat. Cancer Inst. 17, 363 (1956). 8. Weisburger, J. H., Grantham, P. H., Horton, R. E., and Weisburger, E. K., B;ochem. Pharmacol. 19, 151 (1970). 9. Irving, C. C., Xenobioricu 1, 387 (1971). 10. Gutmann, H. R., Anal. Eiochem, 58, 469 (1974). 11. Weisburger, E. K., and Weisburger, J. H., J. Org. Chem. 19, 964 (1954). 12. Weisburger, E. K, and Weisburger, J. H., J. Org. C/rem. 20, 1396 (1955).
MEDICINE
16, 95-103 (1976)
Determination of 2-Acetylaminofluorene and Its Metabolites Urine by High Pressure Liquid Chromatography’ FLOYD Department of Health, The National Center
R. FULLERTON Education and for Toxicological
in
AND C. D. JACKSON Welfare, Food and Drug Administration, Research. Jefferson, Arkansas 72079
Received April 20, 1976
2-Acetylaminofluorene (2-AAF)2 has been used as a model chemical carcinogen since its tumorigenic activity was discovered by Wilson et al. (1) in 1941.2-AAF has been shown to produce tumors in numerous tissues of the rat including liver, Zymbal’s gland, and mammary tissue (l-3) and is known to be a potent bladder carcinogen in the mouse (4). 2-AAF is inactive as a carcinogen per se and requires metabolic activation to the N-hydroxy metabolite (5). The metabolism and reactivity of N-OH-AAF was recently reviewed by Weisburger and Weisburger (6). The other major hydroxy metabolites found in the urine following administration of 2-AAF are I-OH-AAF, 3-OH-AAF, SOH-AAF, and 7-OH-AAF (7,8). Earlier studies on the urinary excretion of 2-AAF and its metabolites utilized paper, silicic acid column, or thin-layer chromatography (7,s). These methods of fractionating and quantitating the various metabolites are time consuming and lack the desired sensitivity for detailed metabolism studies. Recently, Irving (9) reported that N-hydroxy-N-arylacetamides could be analyzed by gas-liquid chromatography as their trimethylsilyl derivatives. Subsequently, Gutmann (10) reported a method utilizing high pressure liquid chromatography for fractionating 2-AAF and some of its hydroxylated metabolites formed by an in vitro microsomal system. We have investigated both gas chromatography and high pressure liquid chromatography as possible methods for analyzing urine samples for 2-AAF and its hydroxy metabolites. It was found that ether extracts of urine contained interfacing materials that precluded the application of the ’ This paper was presented, in part, at the Thirteenth Annual Meeting of the Society of Toxicology, Washington, D.C., March 12, 1974. ’ Abbreviations used: 2-AAF, 2-acetylaminofluorene (Chem. Abstr. nomenclature, N-2fluorenylacetamide); 2-AF, 2-aminofluorene: N-OH-AAF, N-hydroxy-2-acetylaminofluorene; I-OH-AAF, 3-OH-AAF, S-OH-AAF, and 7-OH-AAF, the respective ring-hydroxy derivatives of 2-AAF. 95 Copyright All rights
@ 1976 by Academic Press. Inc. of reproduction in any form resewed.
96
FULLERTON
AND
JACKSON
published gas or liquid chromatographic procedures to the analvsis of urine samples. There was, therefore, a need to modify the existing methods or to develop a new one to permit the analysis of urine samples. This report presents the results of a study in which a high pressure liquid chromatographic method was developed to identify and quantitate 2-AAF and its major metabolites in urine samples. MATERIALS
AND METHODS
Standards of l-OH-AAF, 3-OH-AAF, S-OH-AAF, 7-OHAAF, N-OH-AAF, and 2-AAF were obtained from Dr. Charles Irving, Veterans Administration Hospital, Memphis, Tennessee. Identity and purity of each standard was confirmed by uv spectroscopy (11,12) and thin-layer and paper chromatography (8). The diethyl ether, acetonitrile and heptane were Nanograde, all-glass-distilled solvents from Mallinckrodt Chemical Works, St. Louis, Missouri. The ether and heptane were stored over barium oxide prior to use. Isolation of urinary metabolites. Urinary metabolites were isolated as described by Weisburger et al. (8). Urine samples were mixed with l/10 volume of 1 M sodium acetate, pH 6, and extracted with ether to remove 2-AAF and unconjugated metabolites. The aqueous layer was incubated with fi-glucuronidase (400 units/ml) for 2 hr at 37°C to hydrolyze glucuronide conjugates. Metabolites freed by the P-glucuronidase were extracted with ether and combined with the previous extract. For the purpose of this report, the sulfate conjugates remaining in the aqueous layer were discarded. The ether was evaporated with nitrogen, and the residue was redissolved in the mobile phase described for liquid chromatography. Liquid chromatography. A Waters 2021401 chromatograph equipped with two Model 6000 pumps and a Model 660 solvent programmer was used. The detector utilized a uv photometer operated at 280 nm. Two high pressure liquid chromatography systems were used. The first was essentially the same as described by Gutmann (10). Samples dissolved in 10% heptane in diethyl ether were injected into a column (4 ft x 2-mm i.d.) packed with Carbowax 400 on Porasil C (Waters Associates, Milford, Massachusetts). Fractions were then eluted with 10% heptane in diethyl ether at a rate of l-2 ml/min. The second system consisted of a column (2 ft x 2-mm i.d.) packed with C,,/Corasil (Waters Associates) with a mobile phase of acetonitrile:water using a step-flow gradient. For the first 4 min, a flow of 0.3 mI/min of 12% acetonitrile in water was maintained. After 4 min, a second pump was engaged which added 0.3 ml/min of 60% acetonitrile in water to the initial flow. Chemicals.
CHROMATOGRAPHY
OF Z-AAF METABOLITES
97
RESULTS Liquid Chromatography
Fractionation of 2-AAF and its hydroxy metabolites on a 4-ft Carbowax 400 column is shown in Fig. 1. Each metabolite was identified by comparison of the retention time with that of a corresponding standard. Our results confirmed Gutmann’s report (10) in that N-OH-AAF, SOH-AAF, and 7-OH-AAF were each separated on the Carbowax 400 column. However, we found I-OH-AAF and 3-OH-AAF to have approximately the same retention time as 2-AAF on this column. Although separation of SOH-AAF and 7-OH-AAF was incomplete on the 4-ft column, they could be separated by using a longer column. A more efficient separation of 2-AAF and the ring-hydroxy metabolites by liquid chromatography is shown in Fig. 2. 2-AAF, I-OH-AAF, 3-OH-AAF, S-OH-AAF, and 7-OH-AAF were each well resolved by reverse phase liquid chromatography on a 2-ft column of C&orasil, using acetonitrile:water as the mobile phase. N-OH-AAF, however, was not eluted from this column. The linearity of response (peak height x attenuation) of the chromato-
t
0
:
:
4
:
o
li
TIME
:
’
li (MU)
:
:
16
:
I
20
FIG. 1. Elution profile of 2-AAF and hydroxy metabolites separated by liquid chromatography on a 4-ft x 2-mm i.d. column packed with Carbowax 400. The mobile phase was 10% heptane in diethyl ether with a flow rate of 1.3 mUmin.
98
FULLERTON
AND JACKSON
; Y P--f-i-+ 0
4
! I
: : II 16 TIME (MN)
:
: 20
: 44
FIG. 2. Elution profile of 2-AAF and hydroxy metabolites separated by liquid chromatography using a 2-ft by 2-mm i.d. stainless-steel column packed with C,&orasil. The mobile phase was acetonitrile:water using a step-flow gradient. For the first 4 min, a flow of 0.3 ml/min of 12:88 acetonitrile:water was maintained. At 4 min, a second pump was engaged which added 0.3 mUmin of 60140 acetonitrile:water to the initial flow.
graphic system to the various metabolites is shown in Fig. 3. The response to N-OH-AAF on the Carbowax 400 column was linear from approximately 100 ng to 4.0 pg, while the response to 2-AAF on the C,,/Corasil column was linear from 12 ng to 2 pg (Fig. 3a). Figure 3b shows that the response to each of the ring-hydroxylated metabolites was linear from approximately 10 ng to 2 pg. 5-OH-AAF produced the same response as ‘I-OH-AAF while the response to l-OH-AAF and 3-OH-AAF were identical. Analysis of Urine Samples
Samples of mouse urine containing standards were extracted as described above and the extracts were fractionated by liquid chromatography. N-OH-AAF was eluted from the Carbowax 400 column in fractions relatively free of interfering urinary components and could be quantitated directly from the Carbowax 400 column. There were, however, interfering urinary components when ether extracts of urine were run on C,,/Corasil column. Figure 4a shows the elution profile of an ether extract of control urine chromatographed on the C,,/Corasil column. Numerous components were present which would interfere with iden-
CHROMATOGRAPHY
OF 2-AAF METABOLITES
99
FIG. 3. Linearity of response obtained by liquid chromatography expressed as peak height (absorbance at 280 nm) vs sample size (nanograms). (a) N-OH-AAF determined on a Carbowax 400 column; 2-AAF determined on C,,/Corasil column. (b) Ring-hydroxy derivatives determined on a C,$Corasil column.
tification and quantitation of 2-AAF and the ring-hydroxy metabolites. Subsequently, it was found that the urinary components had markedly different retention times on Carbowax 400 and C,,/Corasil columns. An ether extract of control urine was fractionated on a Carbowax 400 column and fractions corresponding to retention times of 2-AAF and the ringhydroxylated metabolites (8-18 min, Fig. 1) were pooled, concentrated, and rechromatographed on the C,,/Corasil column. The results, shown in Fig. 4b, indicated that the urinary components normally eluted from the C&orasil column were largely removed by a prior fractionation on a
100
FULLERTON
AND JACKSON
FIG. 4. Liquid chromatography of mouse urine components on a C,,/Corasil column. (a) Ether extract of control mouse urine chromatographed on a C,,/Corasil column with no previous cleanup. (b) Same extract was run on a Carbowax 400 column and fractions corresponding to 8 to 18 min (Fig. 2) were concentrated and run on the C&orasil column.
Carbowax 400 column. A combination of the two columns thus provided a method for resolving and quantitating each of the compounds. In a routine analysis, an ether extract of urine was chromatographed on the Carbowax 400 column, and the N-OH-AAF was determined directly from the elution profile. Fractions containing 2-AAF and the ring-hydroxy metabolites were collected, concentrated, and chromatographed on the C&orasil column. Each metabolite was then determined from the elution profile. An analysis of an actual urine sample is shown in Fig. 5. Female BALB/c mice were maintained on a diet containing 0.05% AAF for 2 weeks, and urine was collected overnight in a metabolism cage. Urinary metabolites were isolated as described above. The ether extract was concentrated and fractionated on the Carbowax 400 column (Fig. Sa). Fractions eluting between 13 and 26 min were concentrated and chromatographed on the C,,/Corasil column (Fig. 5b). The quantity of each metabolite was determined by comparing its peak height with that of a standard. The recovery of each metabolite from urine was determined. A mixture containing 200-400 ng of each metabolite was added per milliliter of control urine and processed as described in Materials and Methods. The results are presented in Table 1. The recovery of N-OH-AAF was determined from the Carbowax 400 column while that of 2-AAF and the ring-hydroxy metabolites were determined from the C,,/Corasil column.
CHROMATOGRAPHY
OF 2-AAF METABOLITES
101
FIG. 5. Liquid chromatography profiles of ether extracts of urine from female BALBlc mice maintained on a diet containing 0.05% 2-AAF for 2 weeks. (a) Profile obtained on Carbowax 400 column. (b) Profile of fractions 13 to 26 min from Carbowax 400 column concentrated and run on C,,/Corasil column. Conditions of chromatography described in Materials and Methods.
Recovery of N-OH-AAF proved to be low; approximately 20% was consistently lost during the extraction and/or chromatography procedures. DISCUSSION 2-AAF has been used not only as a model chemical carcinogen but is presently used as a positive control in numerous laboratories in the bioassay of potential carcinogens. Therefore, there is a need for a rapid, sensitive analytical method for determining the amounts of 2-AAF and its major metabolites in urine. Our investigations indicated that the gas chromatographic method described by Irving (9) provided an excellent method for analyzing mixtures of 2-AAF and its hydroxy metabolites when isolated from a relatively “clean” system, but was not applicable to urine samples because of interfering urinary components. Gutmann (10) reported that 2-AAF, N-OH-AAF, S-OH-AAF, and 7-OH-AAF were resolved by liquid chromatography on Carbowax 400 columns, but that l-OH-AAF and 3-OH-AAF had the same retention time as the parent compound 2-AAF. Our results (Fig. 1) confirm his report. However, when the method was applied to ether extracts of urine, numerous urinary components were present which interfered with the determination of several 2-AAF metabolites. Further studies indicated,
102
FULLERTON
AND JACKSON
TABLE RECOVERIES
OF
2-AAF
WHEN
AND
ANAI.YZED
HYDROXY
I METABOLITES
BY LIQUID
Metabolite
FROM
Moust-
URINE
CHROMATOGRAPH\
Percentage recovered”
2-AAF l-OH-AAF 3-OH-AAF .5-OH-AAF 7-OH-AAF N-OH-AAF
101 2 90 k 100 k 112 2 114 k 79 +
2.50 7.3 8.5 6.3 9.3 3.7
Metabolites were added to control urine at concentrations of 200-600 rig/ml. Recoveries of 2-AAF and ring-hydroxy metabolites were determined from a C,,/Corasil column, while recovery of N-OH-AAF was determined from a Carbowax 400 column. * r SD, N = 4.
however, that the interfering urinary components &d different retention times on Carbowax 400 and C,,/Corasil solumns. A combination of the two columns was then found to provide an excellent method for resolving and quantitating 2-AAF, N-OH-AAF, l-OH-AAF, 3-OH-AAF, S-OHAAF, and 7-OH-AAF in the presence of urinary components. The response of the system to each of the metabolites was linear over a range of 10 ng to 2 lug with the exception of N-OH-AAF .which deviated from linearity below 100 ng (Fig. 3). Recoveries of each metabolite from urine were good except for N-OH-AAF. Approximately 20% of this metabolite was consistently lost during the analytical process. This procedure provides a rapid and sensitive analytical method for determining the amounts of 2-AAF and its major metabolites in urine of experimental animals. SUMMARY
2-Acetylaminofluorene, a well-documented carcinogen, is used extensively as a positive control in the bioassay of numerous suspect carcinogens. Consequently, there is a need for a rapid, sensitive analytical method for determining 2-acetylaminofluorene and its metabolites in urine. Such a method would be useful in monitoring workers for potential exposure as well as for determining the applicability of the test species by metabolism studies. A high pressure liquid chromatography method for the identification and quantitation of 2-acetylaminofluorene and its major metabolites in urine has been developed. The primary obstacle in developing the method was the presence of large amounts of endogenous urinary components in ether extracts of urine having chromatographic properties similar to
CHROMATOGRAPHY
OF 2-AAF METABOLITES
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2-acetylaminofluorene and its metabolites. This problem was obviated by the use of a combination of Carbowax 400 and C,,/Corasil columns. N-Hydroxy-2-acetylaminofluorene was determined directly from the Carbowax 400 column. Fractions containing 2-acetylaminofluorene and its l-, 3-, 5, and 7-hydroxy metabohtes were then separated from interfering materials and quantitated by fractionation on a C,,/Corasil column. The response of the system was linear down to 100 ng of N-hydroxy-2acetylaminofluorene and to 10 ng of 2-acetylaminofluorene and the other metabolites. REFERENCES 1. Wilson, R. H., De Eds, F., and Cox, A. J., Cancer Res. 1, 595 (1941). 2. Irving, C. C., Janss, D. H., and Russell, L. T., Cancer Res. 31, 387 (1971). 3. Gutmann, H. R., Malejka-Giganti, D., Barry, E. J., and Rydell, R. E., Cancer Res. 32, 1554 (1972). 4. Wood, M., Eur. J. Cancer $41 (1969). 5. Miller, J. A., and Miller, E. C., Progr. Exp. Tumor Res. 11, 273 (1969). 6. Weisburger, J. H., and Weisburger. E. K., Pharmacol. Rev. 25, 1 (1973). 7. Weisburger, J. H., Weisburger, E. K.. Morris, H. P., and Sober, H. A., J. Nat. Cancer Inst. 17, 363 (1956). 8. Weisburger, J. H., Grantham, P. H., Horton, R. E., and Weisburger, E. K., B;ochem. Pharmacol. 19, 151 (1970). 9. Irving, C. C., Xenobioricu 1, 387 (1971). 10. Gutmann, H. R., Anal. Eiochem, 58, 469 (1974). 11. Weisburger, E. K., and Weisburger, J. H., J. Org. Chem. 19, 964 (1954). 12. Weisburger, E. K, and Weisburger, J. H., J. Org. C/rem. 20, 1396 (1955).